The Alpha Subunit of Hemoglobin Interacts with Endothelial Nitric Oxide Synthase to Regulate Feedback Vasodilation in Human Resistance Arteries

Abstract

Alpha globin, but not beta globin, is expressed in mouse resistance arteries where it binds endothelial nitric oxide (NO) synthase (eNOS) and limits NO signaling. To understand this signaling pathway in humans, we studied omental resistance arteries. Omental tissue was collected during clinically indicated surgery on protocol 13-C-0176 and transferred to the lab on ice. Human resistance arteries 80 to 200 um in diameter were microdissected, cannulated, and perfused with cold PBS to expel blood. We measured gene expression of HBA1 (2658 ± 1190 gene transcripts per 1 ng mRNA ± SEM), HBA2 (3319 ± 1667), HBB (1831 ± 725), and NOS3 (408 ± 49) using RT-ddPCR in human arteries obtained from 7 different subjects. Expression of SLC4A1, a control for blood contamination, was nearly undetectable at (4.2 ± 2.3). To determine the binding partners of vascular alpha globin, protein lysates from omental arteries from three donors were subjected to co-immunoprecipitation with an anti-alpha globin antibody. Western blot revealed both eNOS and beta globin to co-immunoprecipitate (co-IP) with alpha globin. Multiphoton imaging of perfused omental arteries revealed co-localization of alpha globin, beta globin and eNOS to distinct punctates within the plane of the internal elastic lamina (IEL). Given the broadly autofluorescent nature of these complexes (similar to hemoglobin in red blood cells), we used fluorescence lifetime imaging microscopy (FLIM) to identify distinct peak lifetimes of fluorophores labeling antibodies against each of alpha globin, beta globin, and eNOS in the complex. To interrogate the function of endothelial hemoglobin, we performed ex vivo pressure myography on omental arteries obtained from five donors. Maximal vasoconstriction of omental arteries to the alpha-1-adrenergic agonist phenylephrine (PE) was 40.8 ± 3.1% of baseline diameter. Incubation with an alpha globin mimetic peptide that disrupts binding of alpha globin to eNOS, reduced maximal constriction in response to PE to 65.9 ± 2.7% of baseline (p < 0.0001 compared to PE alone), whereas a control peptide had little effect (46.5 ± 7.6% of baseline). Inhibition of eNOS by L-NAME in mimetic-peptide treated arteries restored PE-induced vasoconstriction (44.2 ± 2.3% of baseline). We identified expression of not only alpha but also beta globin in perfused human omental arteries. Co-IP and multiphoton microscopy identified alpha globin, beta globin, and eNOS as components of a complex that localizes near the IEL, likely in myoendothelial junctions. Disruption of alpha globin-eNOS binding enhanced feedback vasodilation to phenylephrine in an NO synthase-dependent fashion. In conclusion, we report that hemoglobin is expressed in human omental arteries where it binds eNOS and regulates alpha-1-adrenergic feedback vasodilation by limiting nitric oxide signaling.